PVS-RIPO is a chimeric poliovirus (PV), consisting of the live attenuated, oral type-1 Sabin vaccine strain carrying a foreign internal ribosome entry site (IRES) from human rhinovirus type 2 (HRV2) (Brown et al., (2014) Cancer 120:3277-86; Goetz et al., (2011) Future Virol. 9:1045-1058; Goetz & Gromeier (2010) Cytokine & Growth Fact Rev; 21:197-203). Poliovirus is the causative agent of Poliomyelitis, a severe neuro-degenerative disease. PVS-RIPO was engineered to be a non-pathogenic therapeutic virus for the treatment of Glioblastoma Multiforme (GBM) and other CD155+ cancers (WO 2014/081937).
The IRES is a critical non-coding sequence element within the 5′ untranslated region of picornaviral genomic RNA, as it mediates translation initiation in a 5′ end-, 7-methyl-guanosine (‘cap’)-independent fashion (Dobrikova et al., (2003) PNAS USA 100:15125-15130; Dobrikova et al., (2003) Virology 311:241-253; Dufresne et al., (2002) J Virol 76:8966-8972). The IRES is the principal determinant of the neuro-attenuated phenotype of all three PV Sabin vaccine strains and of PVSRIPO (Campbell et al. (2005) J Virol 79:6281-6290; Dobrikova et al. (2006) J Virol 80:3310-3321; Brown & Gromeier (2015) Curr Opinion Virol 13:81-85). Mechanistically, neuro-attenuation results from formation of neuron-specific (IRES) ribonucleoprotein complexes that preclude ribosome recruitment. In the Sabin vaccine strains, neuronal IRES incompetence rests on single point mutations mapping to a discrete region of stem loop domain 5; this scant base for neuro-attenuation is the likely cause for their notorious propensity to revert to neurovirulence. Back-reversion can occur through random genetic mutation or homologous recombination with wild-type sequences either during vaccine manufacture or, if live virus is used, following patient administration. PVSRIPO features an intact and functionally integrated HRV2 IRES, a naturally occurring enterovirus. In contrast to the Sabin type 1/3 IRESs, whose deficits were selected for in tissue culture, the HRV2 IRES is inherently neuron-incompetent to a far greater extent than the Sabin IRESs. The genetic footprint for neuronal incompetence in the HRV2 IRES spans at least the upper parts of stem loop domain 5 (˜40 nt) and domain 6 (˜25 nt) (Brown et al., (2014) Cancer 120:3277-86; Dobrikova et al., (2003) PNAS USA 100:15125-15130; Dobrikova et al., (2003) Virology 311:241-253; Dufresne et al., (2002) J Virol 76:8966-8972). Because the consensus HRV2 IRES sequence likely resulted from thousands of years of evolution in humans, it may represent an idealized version of a translation initiation mediator in HRV2 target cells (mitotically active cells in respiratory tract epithelium).
Since polioviruses and PVS-RIPO are single-stranded RNA (ssRNA) viruses, the natural frequency of mutation during viral replication (such as during construct development and manufacturing) is higher than observed with dsDNA viruses and organisms with DNA genomes. Currently, each clinical lot of live or inactivated poliovirus virus must be validated prior to clinical release by in vivo primate neurotoxicity safety testing and in vitro plaque-sequencing methods to verify that the lot does not contain rare genetic reversion mutations that can regenerate a neurotoxic phenotype leading to poliomyelitis. Primate neurotoxicity testing of vaccine lots typically requires several months to complete and in vitro plaque-sequencing methods have relatively poor sensitivity.
Because of their prominent role in the neuro-attenuation of PVs, most of the methods described in the literature for assessing potential polio vaccine neurotoxicity focus on well-defined sites in the IRES (Martin et al., (2011) WHO working group discussion on revision of the WHO recommendation for the production and control of poliomyelitis vaccines (oral): TRS Nos. 904 and 910. Report on Meeting held on 20-22 Jul. 2010, Geneva, Switzerland. Vaccine; 29:6432-6436; Rezapkin et al., (1998) Virology 245:183-187; Chumakov et al., (1994) J Med Virol 42:79-85; Laassri et al., (2006) J Infect Diseases 193:1344-1349). The complete replacement of the polio IRES with the HRV2 IRES in PVSRIPO presents the problem of how to characterize the toxicology profile of the chimeric virus. Since the in vitro methods utilized by polio vaccine manufacturers are not relevant to the HRV2 IRES sequence (i.e., MAPREC), this leaves in vivo test methods and characterization of viral genetic stability as the arbiters of product safety. Primate neurovirulence testing in vivo, including post-administration histology, was performed using cynomolgus macaques following WHO guidelines (Dobrikova et al., (2012) J Virol 86:2750-2759). A prior HTB-15 cell xenograft (in Balb/c mice) and plaque sequencing study demonstrated that the PVSRIPO genome was stable and non-pathogenic after serial passaging and viral expansion in vivo. Using Sanger sequencing, only two polymorphic sites were noted in the HTB-15 xenograft study as positions 97 and 1824 (Dobrikova et al., (2008) Mol Ther 16:1865-1872; Cello J et al., (2008) J Med Virol 80:352-359). Interestingly, neither of these two sites were identified as polymorphic in the Illumina deep sequencing with PVSRIPO, indicating they were the result of de novo base mutations following passaging in HTB-15 cells. Taken together, results of the two prior studies demonstrated the lack of toxicity of PVSRIPO relative to wild-type PV. These two general approaches have been used to establish that PVSRIPO does not exhibit a neuropathic phenotype in primates and, using both direct and plaque-based Sanger sequencing methods, PVSRIPO exhibits a low level of sequence heterogeneity. Other less sensitive methods that make a gross assessment of viral genetic stability and uniformity were also employed such as temperature sensitivity (i.e., RCT40) and general safety testing (e.g., in vivo adventitious agent testing and RT-qPCR). As sequencing technology has progressed, the inherent sensitivity limitation with Sanger sequencing (LoD of ˜15-20% variation per base) has been overcome with ‘Next Generation Sequencing’ (NGS) methods that are capable of <1% theoretical sensitivity to base variants (Cabannes et al., (2014) PDA J Pharm Sci and Tech 68:631-638; Liu et al., (2012) Comparison of next-generation sequencing systems. J Biomed Biotech 2012; 2012:251364; Neverov &Chumakov (2010) PNAS USA 107:20063-20068). Most NGS deep sequencing methods are only limited in sensitivity by their intrinsic mis-incorporation or mis-call error rates which are greater than the sensitivity limits of the underlying base detection technologies.
What is needed are improved methods for detecting mutations in lots of virus-derived therapeutics, such as PVS-RIPO.